From sequence of tumor liberated protein (TLP) to potential targets for diagnosis and therapy

A preliminary analysis of immunoprecipitation followed by Western Blotting (WB) shows corin and TLP precipitate at the same level (approximately 50 KDa) and are recognized by the same antibodies. In parallel the tests of immunoprecipitation were improved by the use of cell extracts derived from lung cancer cells A549 and NCI-H23 with the aim of obtaining a precipitate containing only the TLP. In fact the partial amino acid sequence of TLP shows a high homology with the sequence of human corin (only one amino acid is different) and is present in lung cancer under different isoforms. It is known that human corin is expressed mostly outside the cells and the protein extract derived from the extracellular medium and from the cells transfected with the plasmid, which overexpresses corin, shows several bands analysed on SDS-PAGE that are equivalent to the bands (about 50-100 KDa) observed in the WB analysed by anti-TLP. Introduction While surgery, radiotherapy and chemotherapy are able to cure many cancers, new approaches are required to improve radical curative therapy. A possible route is to utilize the latest achievements made in research on the immunology and genetics of cancer [1]. Cancer immunotherapy [2], or the manipulation of the naturally occurring oncolytic immune reaction, is based on the observation that neoplastic cell antigens stimulate the onset of specific humoral and cellular antibodies both in animals and humans [3]. Certain difficulties that have been encountered reflect the lack of well-purified antigens and/or their ability to unblock cell immunity in the cancer patient. Two ways are known to enhance the host's immunity: aspecific activation (BCG in primis) and specific activation (to stimulate oncolytic circulating and cell antibodies). Moreover, some researchers have performed therapeutic trials with antigens, from autologous and homologous human cancer cells, obtained by various purification procedures [4,5]. The first observation by Tarro et al. [6] demonstrated that when TLP is extracted from a tumor, purified in the laboratory, and reintroduced into the patients body, it boosts the immune system’s cancer responsive capabilities [7]. As lung cancer accounts for the largest number of cancer deaths in the Western world, TLP may have the potential to greatly improve the cure rate and or serve as a lung cancer vaccine (Table 1) [8]. Corin is a cardiac serine protease that activates natriuretic peptides. It consists of an N-terminal cytoplasmic tail, a transmembrane domain, and an extracellular region with a C-terminal trypsin-like protease domain. The transmembrane domain anchors corin on the surface of cardiomyocytes. To date, the function of the corin cytoplasmic tail remains unknown [9]. Corin shows high homology with TLP and is present in various isoforms in the lung [10. If the fragments cut with thrombin proved to be the same, the data would support the hypothesis that TLP and corin are the same protein. At the same time a plasmid was used that allows us to transfect and over-express human corin with the purpose of assessing whether the two proteins are actually the same protein or are different by Western blotting (using anti-TLP and anticorin antibodies). Materials and methods Antigen synthesis and antibody production The synthesis of the TLP-derived peptide RTNKEASI [23] and two different rabbit polyclonal anti-RTNKEASI immune sera were carried out at Rockland Immunochemicals Inc. (Gilbertsville, PA, USA) and at Biogenes GmbH (Berlin, Germany). The serum anti-TLP produced at Rockland was further purified against RTNKEASI peptide by chromatography. Cell culture The human cell lines A549, CA46, HL60, MCF7, MRC-5, Hela and PC3 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). A549, MRC-5, HL60, Hela, and MCF7 cell lines were cultured in Glutamax Dulbecco’s Modified Eagle’s Correspondence to: Giulio Tarro, Via Posillipo 286, 80123 Naples, Italy; E-mail: [email protected]; [email protected]